1. Field of the Invention
This invention relates to the purification and separation of moieties, particularly those of biological interest, from mixtures containing them utilizing improved tangential-flow filtration processes and apparati.
2. Description of Related Disclosures
Several methods are currently available to separate molecules of biological interest, such as proteins, from mixtures thereof. One important such technique is affinity chromatography, which separates on the basis of specific and selective binding of the desired molecules to an affinity matrix or gel. Affinity gels typically consist of a ligand-binding moiety immobilized on a gel support. For example, GB 2,178,742 utilizes affinity chromatography to purify hemoglobin and its chemically modified derivatives based on the fact that native (oxy)hemoglobin binds specifically to polyanionic moieties of certain affinity gels. In this process, unmodified hemoglobin is retained by the affinity gel, while modified hemoglobin, which cannot bind to the gel because its polyanion binding site is covalently occupied by the modifying agent, is eluted. Affinity chromatography columns are highly specific and thus yield very pure products; however, affinity chromatography is a relatively expensive process.
Another known separation method is membrane filtration, which separates dissolved and suspended solutes on the basis of their size. In the simplest form of this process, a solution is forced under pressure through a filter membrane with pores of a defined size. Solutes larger than the pore size of the membrane filter are retained, while smaller solutes are carried convectively through the membrane with the solvent.
Such membrane filtration processes generally fall within the categories of reverse osmosis, ultrafiltration, and microfiltration, depending on the pore size of the membrane. Conventionally, ultrafiltration employs membranes rated for retaining solutes between approximately 1 and 1000 kDa in molecular weight, reverse osmosis employs membranes capable of retaining salts and other low molecular weight solutes, and microfiltration, or microporous filtration, employs membranes in the 0.1 to 10 micrometer (micron) pore size range, typically used to retain colloids and microorganisms.
Over the past 25 years, ultrafiltration has progressed from a small-scale laboratory tool to a fully established unit operation capable of processing thousands of liters per hour. Ultrafiltration is widely used for protein concentration and removal of salts and alcohols, as well as in depyrogenation of process and rinse water, saline solutions, and low molecular weight additives. The advantages of ultrafiltration include low energy cost, low capital equipment outlay, and efficient and controllable operation with very low denaturation of product.
However, limitations exist on the degree of protein purification achievable in ultrafiltration. These limits are due mainly to the phenomena of concentration polarization, fouling, and wide membrane pore size distribution. Hence, solute discrimination is poor. See, e.g., Porter, ed., Handbook Of Industrial Membrane Technology (Noyes Publications, Park Ridge, N.J., 1990), pp. 164-173.
A polarized layer of solutes acts as an additional filter in series with the original ultrafilter, and provides significant resistance to the filtration of solvent. The degree of polarization increases with increasing concentration of retained solute in the feed, and can lead to a number of seemingly anomalous or unpredictable effects in real systems. For example, under highly polarized conditions, filtration rates may increase only slightly with increasing pressure, in contrast to unpolarized conditions, where filtration rates are usually linear with pressure. Use of a more open, higher-flux membrane may not increase the filtration rate, because the polarized layer is providing the limiting resistance to filtration. The situation is further complicated by interactions between retained and eluted solutes.
A result of concentration polarization and fouling processes is the inability to make effective use of the macromolecular fractionation capabilities of ultrafiltration membranes for the large-scale resolution of macromolecular mixtures such as blood plasma proteins See Michaels, "Fifteen Years of Ultrafiltration: Problems and Future Promises of an Adolescent Technology" in Anthony R. Cooper, ed., Ultrafiltration Membranes and Applications, Polymer Science and Technology, 13(Plenum Press, N.Y., 1979), pp. 1-19, at p. 9. Consequently, the potentially exciting utilization of membrane ultrafiltration for large-scale complex macromolecular mixture-separations currently performed by such techniques as gel permeation, adsorption, or ion-exchange chromatography, selective precipitation, or electrophoresis is considered elusive.
The merits of various multi-stage and cascaded cross-flow filtration schemes have been examined on paper, with a slightly improved effect. See Michaels and Matson, Desalination, 53: 231-258 (1985), p. 235 in particular. See also the experimental flow circuit in FIG. 1 on p. 535 of van Reis et al., J. Interf. Res., 2: 533-541(1982), and M. Cheryan, Ultrafiltration Handbook, (Technomic Publishing Co., Inc.: Penn., 1986), p. 311, where a cascade membrane recycle bioreactor system for producing protein hydrolyzate fractions of different molecular sizes is described.
To circumvent the effects of concentration polarization, several processes have been developed that modify the feed plasma source to improve selectivity and flux (defined as the filtration rate divided by the membrane area). For example, UK Appln. No. 2,065,129 describes a separation technique wherein serum is diluted to reduce total protein and salt concentration while the pH is adjusted to between 3.8 and 4.7 prior to ultrafiltration. Baeyer et al., J. Membrane Sci., 22:297-315(1985) describes a process wherein the plasma is first diluted by a factor of 12 prior to ultrafiltration. U.S. Pat. No. 4,350,156 discloses a process for removing macromolecules from plasma by cooling the plasma to about 10.degree. C. and then filtering the macromolecules from the cooled plasma to form a filtered low molecular weight plasma stream. This process does not employ an ultrafiltration membrane.
One major approach to address concentration polarization in ultrafiltration systems has been to control the fluid flow pattern so as to enhance transport of the retained solute away from the membrane surface and back into the bulk of the feed. In a process known as tangential-flow ultrafiltration (TFF), the feed stream is recirculated at high velocities tangential to the plane of the membrane to increase the mass-transfer coefficient for back diffusion. Gabler, ASM News, 50:299(1984). The fluid flowing in a direction parallel to the filter membrane acts to clean the filter surface continuously and prevents clogging by non-filterable solutes. Another filtration device that achieves the same effects as TFF is a rotary filtration device containing an outer and inner cylinder, where the inner cylinder is rotated to create a vortex to obtain high velocity without a change in pressure.
In TFF, a pressure differential gradient, called transmembrane pressure (TMP), is applied along the length of the membrane to cause fluid and filterable solutes to flow through the filter. Flux is independent of TMP above a certain minimum value that can be determined empirically. To achieve maximum flux, ultrafiltration systems are typically run with an outlet pressure equal to or greater than this minimum value. Hence, flux is constant along the length of the membrane, while the TMP varies. Both laminar and turbulent flow approaches have been used with some success; however, conventional TFF still affords only poor molecular size resolution.
Attempts have been made to combine affinity separation with TFF to increase the ability of TFF to separate selectively based on biological differences. See WO 87/04169 published Jul. 16, 1987. In TFF ultrafiltration, a soluble affinity polymer is placed in the mixture containing the fractions to be separated, on the upstream side of the filter membrane. Since the polymer is much larger than the solute particles, a filter can be selected that will allow unbound solutes to pass through the filter but prevent the passage of polymer and any substance bound to the polymer. Due to the problems posed by conventional TFF, however, even this approach has met with limited success.
U.S. Pat. No. 4,105,547 issued Aug. 8, 1978 discloses a filtering process, designed especially for ultrafiltration, in which a filterable fluid is caused to flow under pressure through a filtering passage extending along one side of a filter, in such a way that a considerable pressure drop arises along the filter area in the flow direction. In the apparatus employed, the pressure difference between both sides of the filter is maintained substantially constant throughout the entire filter area. The patentees teach that clogging of the filter tending to reduce the flow can be compensated for by successively raising the driving pressure to a level that is below the pressure-independent region of the flux v. TMP curve. This raising of the driving pressure results in constant filtration rate, but not higher selectivity. In addition, the patentees disclose that the transmembrane pressure should be increased as the filtration is being carried out. Other features taught by the patentees include better membrane cleaning.
U.S. Pat. No. 4,191,182 issued Mar. 4, 1980 discloses in one embodiment a process and apparatus for continuously separating blood into plasma and cellular component fractions. The process involves withdrawing whole blood and pumping it into a filtering chamber of a filtration cell, and continuously filtering the whole blood by passing it in a flow over and parallel to a membrane of a certain pore size range and at a flow rate sufficient to provide a specified shear stress range at the membrane interface within particular TMP confines. Then, the cellular component fraction is continuously mixed with an amount of replacement fluid substantially equal to the separated plasma fraction, and the cellular component fraction and replacement fluid mixture are continuously returned to a blood vessel of the donor.
In one embodiment a portion of the plasma fraction separated from the whole blood is recycled in a flow parallel to and in the same direction as the flow of the whole blood over the filter membrane, but on the opposite side of the membrane from the flow of whole blood, to obtain a substantially uniform TMP across the entire length of the membrane. The purpose of this embodiment is to increase filtration rates to twice that when plasma filtrate is removed from the filtrate chamber without such recycling. Also, it allows for the use of long filtering chamber flow paths and for the design of coil-type filtration cells.
More recently, U.S. Pat. No. 4,789,482 issued Dec. 6, 1988 describes a process for separating blood plasma into high and low molecular weight streams. The blood plasma is introduced at a certain shear rate into an inlet portion of a separation unit containing several thin channels or hollow fibers with walls through which ultrafiltration is carried out. The high and low molecular weight streams are separated from the unit, the high molecular weight stream is recirculated to an inlet portion of the separation unit to form a recirculation stream, and the ratio of the recirculation stream flow rate to the permeate stream flow rate is controlled between 5 and 100, with the ratio of TMP at outlet to inlet of the separation unit between about 0 and 0.85. This method uses the conventional thinking regarding ultrafiltration, i.e., that the outlet and inlet TMPs must be different; thus, it does not overcome the concentration polarization problem inherent in ultrafiltration.
Despite all of these attempts at improvement, it is still the case that although concentration polarization can be modified, and quite high filtration rates can be achieved even from very concentrated solutions if appropriate flow conditions are supplied, the polarized layer can never be completely eliminated. This is observed from a number of protein mixtures, and a rule of thumb has evolved that fractionation of protein mixtures by simple ultrafiltration will probably be highly inefficient unless the species are at least a factor of ten different in molecular weight. Although some closer separations have been occasionally reported, they have always been under extremely and impractically dilute conditions. Nelsen, "Ultrafiltration in Plasma Fractionation", in Proceedings of the Internat. Workshop on Technology for Protein Separation and Improvement of Blood Plasma Fractionation, Reston Va., Sep. 7-9, 1977, Sandberg, ed., NIH, DHEW Pub. No. NIH 78-1422, p. 137; Flaschel et al, "Ultrafiltration for the Separation of Biocatalysts", in Fiechter, ed., Advances in Biochemical Engineering/Biotechnology, Vol. 26(Downstream Processing), pp. 73-142 (1983), particularly, p. 124; Cheryan, supra, p. 218-219.
It is an object of the present invention to provide tangential-flow filtration processes for separating species such as particles and molecules by size, which processes are selective for the species of interest, resulting in higher-fold purification thereof.
It is another object to provide improved filtration processes, including ultrafiltration processes, for separating biological macromolecules such as proteins which processes minimize concentration polarization and do not increase flux.
It is another object to provide a filtration process that can separate by size species that are less than ten-fold different in size and does not require dilution of the mixture prior to filtration.
These and other objects will become apparent to those skilled in the art.